The Rotor-Gene Multiplex RT-PCR Kit is designed for use with the Rotor-Gene Q and other Rotor-Gene cyclers, providing ultrafast, highly reliable quantification in multiplex, real-time one-step RT-PCR using sequence-specific probes. Depending on the cycler configuration, up to 4 RNA targets (e.g., 1 control gene and 3 target genes) can be quantified simultaneously in the same tube. Outstanding performance is achieved through the combination of a specially optimized master mix and the unique Rotor-Gene cycler. For convenience, the master mix can be stored at 2–8°C.

Duplex, real-time one-step RT-PCR was performed using the Rotor-Gene Multiplex RT-PCR Kit and self-designed TaqMan assays for 28S rRNA (Cyanine 670 dye; data in inset) and PPIA (cyclophilin A, FAM dye). Triplicate reactions were run on the Rotor-Gene 3000 using leukocyte RNA as template (10-fold dilutions from 100 ng to 10 pg). The amplification plots for the duplex reactions (colored) overlapped with those for control singleplex reactions (gray), demonstrating the reliability of the duplex analysis.|[A] Q-Bond in Rotor-Gene Multiplex PCR Master Mix increases the affinity of DNA polymerase for short single-stranded DNA, reducing primer annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing primer annealing time.|Rotor-Gene Multiplex RT-PCR Kits provide a simple procedure for quantitative, multiplex, real-time PCR. In contrast to current methods, the kits eliminate the need for optimization of the concentrations of primers, Mg2+, and Taq DNA polymerase, even for difficult assays (e.g., assays in which the copy number of the target gene is much smaller than that for the reference gene).|[A] NH4+ ions prevent nonspecific primers from annealing to the template. [B] Synthetic Factor MP, an innovative PCR additive, increases the local concentration of primers at the template. Together with K+ and other cations, Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq Plus DNA Polymerase.|Duplex, real-time one-step RT-PCR using in vitro transcripts of [A] HSP90AA1 (109, 107, 105, 103, or 10 copies) and [B] GAPDH (109 copies) was performed (colored curves). For comparison, singleplex RT-PCR was also carried out (gray curves). Duplicate reactions were run on the Rotor-Gene 6000 using the Rotor-Gene Multiplex RT-PCR Kit and self-designed TaqMan assays. Reliable duplex RT-PCR is demonstrated by the evenly spaced curves for HSP90AA1 and overlapping curves for GAPDH. The efficiency of [C] HSP90AA1 amplification was in an optimal range.|4-plex, real-time one-step RT-PCR was performed using the Rotor-Gene Multiplex RT-PCR Kit and self-designed TaqMan assays for the indicated targets. Reactions were run on the Rotor-Gene Q using 100, 10, 1, or 0.1 ng RNA from HeLa cells. The plots of CT value versus log template amount were parallel, indicating all 4 targets were amplified with the same high efficiency. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; RPS27A: ribosomal protein S27a; NFKB: nuclear factor of kappa light polypeptide gene enhancer in B-cells.|

The Rotor-Gene Multiplex RT-PCR Kit reliably quantifies low- to high-abundance targets in duplex, triplex, and 4-plex assays from as little as 10 pg template, and can detect 10 copies of target (see figures "Reliable duplex analysis" and "Efficient, sensitive duplex analysis"). All targets in a multiplex assay are amplified with equally high PCR efficiency (see figure "Highly efficient 4-plex analysis"). This enables reliable relative quantification, where the expression of a target gene is normalized to that of a control gene.

Principle

Amplifying reference and target genes in the same reaction instead of in separate reactions increases the reliability of gene quantification by minimizing handling errors. The Rotor-Gene Multiplex RT-PCR Kit enables reliable multiplex quantification of RNA targets on the Rotor-Gene Q without the need for optimization of reaction and cycling conditions (see flowchart "QIAGEN multiplex kits"). Real-time one-step RT-PCR is carried out, enabling reverse transcription and PCR to take place sequentially in the same reaction vessel. Since it is not necessary to transfer the finished reverse transcription reaction to another tube for PCR, the real-time RT-PCR procedure is streamlined, making high-throughput analysis possible.

Highly specific amplification is assured through a balanced combination of K+ and NH4+ ions, which promote specific primer annealing and enable high PCR specificity and sensitivity, while synthetic Factor MP, an innovative PCR additive specially developed for challenging multiplex PCR applications, allows different amplicons in the same reaction to all be amplified with the same high efficiency (see figure "Unique PCR buffer").

Fast cycling without compromising performance is achieved using Q-Bond, a novel PCR additive that considerably shortens cycler run times (see figure "Fast primer annealing"). In addition, an optimized mix of reverse transcriptases provides efficient cDNA synthesis in just 15 minutes, while the highly stringent hot-start enzyme HotStarTaq Plus DNA Polymerase is rapidly activated at the start of PCR by a brief 5-minute incubation at 95ºC.

Components of 2x Rotor-Gene Multiplex RT-PCR Kit*

Component

Features

Benefits

HotStarTaq Plus DNA Polymerase

5 min activation at 95ºC

Set up of qPCR reactions at room temperature

Rotor-Gene Multiplex RT-PCR Buffer

Balanced combination of NH4+ and K+ ions

Specific primer annealing ensures reliable qPCR results

Synthetic Factor MP

Reliable multiplexing analysis of up to 4 genes in the same tube

Unique Q-Bond additive

Faster PCR run times enable faster results and more reactions per day

Rotor-Gene RT Mix

Special blend of reverse transcriptases with a high affinity for RNA

RNA can be transcribed in just 15 minutes, even through complex secondary structures

* Also contains dNTP mix (dATP, dCTP, dGTP, dTTP).

Procedure

A ready-to-use master mix eliminates the need for optimization of reaction and cycling conditions, such as primer and probe concentrations. Simply add template RNA, primer–probe sets, and the supplied reverse transcriptase mix to the master mix and program the cycler. The handbook supplied with the kit lists recommended dyes and contains a single protocol for all multiplex RT-PCR assays.

Applications

The Rotor-Gene Multiplex RT-PCR Kit is optimized for fast, real-time one-step RT-PCR analysis using sequence-specific probes on the Rotor-Gene Q. It is also compatible with the Rotor-Gene 3000 and the Rotor-Gene 6000. Up to 4 RNA targets can be simultaneously and rapidly quantified in the same tube, increasing throughput and saving precious sample material.